CN113993515A

CN113993515A - Methods of treating solid tumors using crocetin

Info

Publication number: CN113993515A
Application number: CN201980095964.XA
Authority: CN
Inventors: 钱江; 曹浪
Original assignee: Dr Q Laboratory Co ltd; Hangzhou Menglan Ruisi Biotechnology Co ltd
Current assignee: Dr Q Laboratory Co ltd; Hangzhou Menglan Ruisi Biotechnology Co ltd
Priority date: 2019-12-10
Filing date: 2019-12-10
Publication date: 2022-01-28
Anticipated expiration: 2039-12-10
Also published as: WO2021114089A1; CN113993515B

Abstract

Novel uses of crocetin are disclosed. In particular, the present disclosure relates to methods of treating solid tumors, such as glioblastoma, by administering a pharmaceutically effective amount of crocetin to a mammal prior to, during, or after treatment as radiation therapy, chemotherapy, immunotherapy, or a combination thereof. The present disclosure also relates to the use of crocetin in compositions for the treatment of solid tumors, such as glioblastoma.

Description

Methods of treating solid tumors using crocetin

Technical Field

The present disclosure relates to crocetin having therapeutic use in the treatment of solid tumors. For example, the present disclosure relates to methods of treating solid tumors, particularly glioblastoma, by administering a pharmaceutically effective amount of crocetin to a mammal in need thereof, before, during, or after a therapy of radiation therapy, chemotherapy, immunotherapy, or a combination thereof. The present disclosure also relates to the use of crocetin in a composition for the treatment of solid tumors, in particular glioblastoma.

Background

Malignant cancers such as hematological tumors and solid tumors pose a serious threat to human health. Treatment of cancer has traditionally been accomplished by one or a combination of surgery, chemotherapy, radiation therapy, immunotherapy and hormonal therapy. With the emergence of new anticancer drugs, especially the rise of immunotherapy, the curative effect of hematological tumors has been greatly improved in recent years, and radiotherapy and chemotherapy are still the main treatment methods after solid tumor surgery.

However, radiation therapy and chemotherapy have damage or toxicity to normal tissues and organs, resulting in decreased autoimmunity and possibly certain side effects such as alopecia, nausea, vomiting, loss of appetite, impaired immune system, bone marrow suppression, and the like. These side effects may compromise the efficacy of chemotherapy and radiotherapy, and even result in complete cessation of chemotherapy and radiotherapy. Unfortunately, attempts to develop chemotherapeutic drugs that are capable of targeting specific molecules or cancer-associated signaling pathways and reducing side effects often fail to produce the expected improvement in patient prognosis. This is primarily due to the ability of cancer cells to enhance viability using a combination of a number of different cellular mechanisms. In many cases, cancer cells are able to circumvent targeted therapy-induced apoptosis by simply activating other survival pathways after initial therapy. Meanwhile, the treatment effect of chemotherapy is still not ideal due to factors such as primary and acquired drug resistance of tumor cells.

Glioblastoma is the most common primary intracranial tumor, is caused by brain and spinal glial cell canceration, and has the characteristics of high morbidity, high recurrence rate, high mortality and low cure rate. It is not significantly different from normal brain tissue due to its invasive growth. And in most cases it is not limited to only one lobe, penetrating the brain tissue in a finger-like manner, thereby damaging the brain tissue. Glioblastomas account for approximately 12% to 15% of all brain tumors (85% to 90% of all primary Central Nervous System (CNS) tumors). The number of new cases of tumors in the central nervous system in the united states is about 18,800 (6.6 per 100,000) cases per year, and about 12,800 (4.7 per 100,000) deaths. This type of cancer accounts for approximately 1.3% of all cancers in the united states and 2.2% of all cancer-related deaths in the united states. In china, there are over 100,000 new cases per year. Worldwide, there are approximately 176,000 new cases of brain and other central nervous system tumors each year with an estimated mortality rate of 128,000. Generally, primary brain tumors are higher in white races than in black races, and mortality is higher in males than in females. The peak of the disease occurs between the ages of 45 and 70, wherein the incidence rate of children is the highest. Glioblastoma multiforme (GBM) is the most common and deadliest type and remains an incurable disease. Even with the most aggressive treatment regimens, GBM patients have an average life expectancy of only 12-15 months.

The current standard treatment for glioblastoma is maximal surgical resection followed by radiotherapy in combination with Temozolomide (TMZ) chemotherapy (concurrent or post radiotherapy). Specifically, glioblastoma patients received 2Gy fractionated topical irradiation daily for 5 days per week for 6 weeks (60 Gy total), with the addition of temozolomide at 75mg/m per day²On a daily basis, after 1 month of rest, 6 cycles of adjunctive temozolomide (150 to 200 mg/m)²Day, 5 consecutive days in each 28 day cycle). However, the two-year survival rate of patients receiving standard treatment is still low. Therefore, there is an urgent need to develop new effective therapeutic agents and/or effective radiation/chemotherapy sensitizers to treat glioblastoma.

Historically, natural products have been a source of successful pharmaceutically active compounds, especially with fewer side effects in chemotherapy. In fact, 63% of the anticancer drugs used between 1981 and 2006 are natural products, inspired by natural products or synthesized from natural pharmacophores. Pharmaceutically active compounds derived from natural materials have the potential to provide targeted cytotoxic and immunomodulatory responses while limiting the side effects associated with currently used cancer therapies. The use of natural products attempts to balance the powerful capabilities of both multiple approaches with the history of human safe consumption and benign side effects.

Crocetin (C)₂₀H₂₄O₄CAS: 27876-94-4) is a naturally occurring 8,8 '-diapo- ψ, ψ' carotenedioic acid having a wide range of physiological activities. Crocetin is brick red crystal, melting point 285 ℃, and insoluble in water and most organic solvents. Crocin has the following chemical structure:

crocetin has been used for over 1000 years as a secondary compound in safflor yellow or Gardenia yellow, which is a colorant extracted from the fruits of saffron (Crocus salivus) or Gardenia (Gardenia jasminoides). The content of natural crocetin in saffron and gardenia is very rare. Commercially, crocetin is obtained mainly by removing sugar groups after the hydrolysis of crocin. It is now also available by chemical synthesis.

Crocetin has been shown to be effective in increasing oxygen diffusion and oxygen consumption in mammalian tissues. Crocetin is sparingly soluble in water and common solvents, limiting its use in foods, beverages, pharmaceuticals and nutritional supplements. Trans Sodium Crocetin (TSC) is obtained by reacting crocetin with sodium hydroxide to increase its solubility in water (see U.S. patent No.6,060,511). TSC has been shown to treat a range of diseases such as hypertension, atherosclerosis, cardiovascular disease, peripheral vascular disease, stroke, vascular thrombosis, cerebral ischemia, ischemic osteonecrosis, chronic ocular disease, macular degeneration, diabetic retinopathy, and the like.

Crocetin is reported to be ineffective in treating more severe (55% -60%) blood loss. In contrast, TSC performed well in a more severe hemorrhagic shock model (see Gainer, Expert Opinion on Investigational Drugs, 17: 6, 917-924 (2008)). In one study of the effect of TSC on cerebral infarction, a U-shaped reduction in infarct volume was observed: the reduction in infarct volume reached statistical significance in the dose range of 23 to 229. mu.g/kg, with the greatest protective effect at the dose of 92. mu.g/kg. (see Manabe et al, J neurosurg.113 (4): 802-809 (2010)). In contrast, in studies of TSC on the enhanced efficacy of radiotherapy and chemoradiotherapy on survival in the glioblastoma model, it was found that peak efficacy was achieved at doses around 100 μ g/kg (see Sheehan et al, J Neurosurg.108: 972-. A clinical trial was subsequently conducted in the united states to determine the effectiveness of the treatment of GBM by adding TSC (hereinafter "TSC + radiotherapy") during radiotherapy. A phase II clinical outcome was published in 2017, where Kaplan-Meier analysis showed that 36% of patients receiving TSC + radiotherapy survived at 2 years, compared to a historical 2-year survival rate of 27% to 30% for standard treatment methods. This result strongly suggests that the addition of TSC during radiation therapy is beneficial for the treatment of GBM (see Gainer et al, J Neurosurg.126: 460-466 (2017)).

In the treatment of solid tumors, especially glioblastoma, the medical need to develop new therapeutic drugs and/or sensitizers for radiotherapy and chemotherapy is still not met. The inventor surprisingly finds that the curative effect of radiotherapy and chemotherapy can be obviously improved by applying the crocetin before the radiotherapy or the chemotherapy or the combined radiotherapy/chemotherapy, so that the solid tumor can be effectively treated, and the tumor recurrence and metastasis are prevented.

Disclosure of Invention

In one aspect, the present disclosure provides a method of treating a solid tumor in a mammal comprising administering a pharmaceutically effective amount of crocetin to the mammal prior to, during, or after radiation therapy, chemotherapy, immunotherapy, or a combination thereof.

In some embodiments, the mammal is a human.

In some embodiments, the radiation therapy is selected from the form of electromagnetic waves, such as X-rays or gamma rays, or charged or neutral particles.

In some embodiments, the radiation therapy is administered by external irradiation, interstitial implant, or a combination thereof.

In some embodiments, radiation therapy is administered at a dose of about 60-70Gy over 4-7 weeks.

In some embodiments, the solid tumor disclosed herein is selected from glioblastoma, squamous cell carcinoma, skin cancer-related tumors, breast cancer, head and neck cancer, gynecological cancer, urinary and male genital cancer, bladder cancer, prostate cancer, bone cancer, endocrine adenocarcinoma, cancer of the digestive tract, breast cancer, cancer of the major digestive tract/organ, cancer of the central nervous system, and lung cancer.

In another embodiment, the solid tumor is a glioblastoma.

In some embodiments, the chemotherapeutic method is selected from the group consisting of temozolomide, cisplatin, methotrexate, or paclitaxel administered together with the drug.

In another embodiment, the method of chemotherapy is with temozolomide.

In one embodiment, the solid tumor is a glioblastoma and the chemotherapy is administration of temozolomide.

In some embodiments, crocetin is used as a sensitizer.

In some embodiments, the method further comprises administering with at least one anti-cancer method.

The anti-cancer methods disclosed herein are selected from other sensitizers, targeted therapeutics and immunotherapeutics in the treatment of cancer.

In another embodiment, the anti-cancer method is selected from the group consisting of anti-cancer alkylating agents, anti-cancer antimetabolites, anti-cancer antibiotics, plant-derived anti-cancer agents, anti-cancer platinum coordination compounds, anti-cancer camptothecin derivatives, anti-cancer tyrosine kinase inhibitors, monoclonal antibodies, and biological response modifiers.

In some embodiments, the pharmaceutically effective amount of crocetin is administered prior to therapy as radiation therapy, chemotherapy, immunotherapy, or a combination thereof.

In another aspect, the present disclosure provides a composition comprising crocetin and at least one pharmaceutically acceptable carrier or adjuvant for use in the treatment of solid tumors.

In some embodiments, the composition is in the form of an injection, tablet, capsule, pill, suppository, aerosol, oral liquid, granule, powder, sustained release formulation, nano-formulation, syrup, wine, tincture, lotion, film, or combination.

In some embodiments, the composition is in the form of a liposomal formulation.

In some embodiments, the composition is administered orally, by injection, by implantation, by spraying, by inhalation, or a combination thereof.

In another aspect, the present disclosure provides a method of sensitizing a mammal to a therapy of radiation therapy, chemotherapy, or a combination thereof comprising administering to the mammal a pharmaceutically effective amount of crocin.

In some embodiments, the mammal is a human.

Drawings

FIG. 1 shows the relative volume of Hela subcutaneous tumors (Vt/V0) in BALB/c nude mice as a function of the days of observation.

FIG. 2 shows the relative volume of subcutaneous tumors (Vt/V0) in BALB/c nude mice HCT116 as a function of the number of days observed.

FIG. 3 shows the relative volumes of subcutaneous tumors (Vt/V0) of BALB/c nude mice HepG2 as a function of the number of days of observation.

FIG. 4 shows the Kaplan-Meier survival curves of BALB/C nude mouse C6 orthotopic glioma model animals.

FIG. 5 shows the Kaplan-Meier survival curves of BALB/C nude mouse C6 orthotopic glioma model animals.

FIG. 6 shows the Kaplan-Meier survival curves of BALB/C nude mouse C6 orthotopic glioma model animals.

FIG. 7 shows the Kaplan-Meier survival curves of C57BL/6 mouse syngeneic GL261 orthotopic glioma model animals.

FIG. 8 shows the Kaplan-Meier survival curves for C57BL/6 mouse syngeneic GL261 orthotopic glioma model animals.

Detailed Description

Unless otherwise indicated, the following terms are to be understood to have the following meanings:

as used herein, including the claims, the singular forms of words such as "a" (an), an "(an), and the" (the), include their corresponding plural references unless the context clearly dictates otherwise.

As used herein, the term "or combinations thereof refers to all permutations and combinations of the items listed prior to that term. For example, "A, B, C or a combination thereof" is intended to include at least one of: A. b, C, AB, AC, BC, or ABC, if the order is important in a particular context, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, explicitly included are combinations that contain repetitions of one or more items or terms, such as BB, AAA, AB, BBC, aaabccccc, CBBAAA, CABABB, and the like. The skilled artisan will appreciate that there is generally no limitation on the number of items or terms in any combination, unless otherwise apparent from the context.

As used herein, the term "cancer" refers to a proliferative disease caused by or characterized by cellular proliferation that has lost susceptibility to normal growth control. The term "cancer" as used in this application includes tumors and any other proliferative disease. Cancers of the same tissue type originate in the same tissue and may be divided into different subtypes according to their biological characteristics. The cancer may be selected from, for example, glioblastoma, squamous cell carcinoma, skin cancer-related tumors, breast cancer, head and neck cancer, gynecological cancer, urinary and male genital cancer, bladder cancer, prostate cancer, bone cancer, endocrine adenocarcinoma, cancer of the digestive tract, cancer of the main digestive tract/organ, cancer of the central nervous system, and lung cancer.

Glioblastoma, also known as glioblastoma multiforme, may develop from diffuse or anaplastic astrocytoma, but is more commonly a new-onset, precursor without evidence of less malignancy. Histologically, this tumor is an anaplastic cytoglioma consisting of poorly differentiated, usually pleomorphic astrocytic tumor cells with marked nuclear atypia and active mitotic activity. Glioblastoma predominantly affects the cerebral hemisphere. For example, central nervous system tumors are associated with altered oncogenes, altered tumor suppressor genes and characteristic patterns of chromosomal abnormalities.

Skin cancer-associated tumors include squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merck's cell skin cancer, and non-melanoma skin cancer. Head and neck cancers include, for example, laryngeal, hypopharyngeal, nasopharyngeal, oropharyngeal cancers, lip and oral cancers, as well as squamous cell carcinomas. Lymphomas include, for example, AIDS-related lymphoma, non-hodgkin's lymphoma, cutaneous T-cell lymphoma, burkitt's lymphoma, hodgkin's disease, and central nervous system lymphoma.

Examples of breast cancer include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ. Examples of cancers of the respiratory tract include, but are not limited to, small cell and non-small cell lung cancers, as well as bronchial adenomas and pleural pneumococcal tumors.

Examples of urinary and male genital cancers include, but are not limited to, bladder cancer, penile cancer, kidney cancer, renal pelvis cancer, ureter cancer, urethral cancer, testicular cancer, and human papillary renal cancer.

Examples of gynecological cancers include, but are not limited to, endometrial, cervical, ovarian, vaginal, and vulvar cancers, as well as uterine sarcomas.

Endocrine adenocarcinomas can be named by reference to the hormones they produce, for example, gastrinomas (producing gastrin), insulinomas (producing insulin), somatostatinoma (producing somatostatin), VIPomas (producing VIP) and glucagonomas (producing glucagons)

Examples of digestive tract cancers include, but are not limited to, anal, colon, colorectal, esophageal, gallbladder, gastric, pancreatic, rectal, small intestine, and salivary gland cancers.

Examples of lung cancer include, but are not limited to, small cell lung cancer and non-small cell lung cancer, including squamous cell lung cancer, adenocarcinoma, and large cell lung cancer.

These diseases have been well characterized in humans, but similar etiologies exist in other mammals and can be treated by administering the pharmaceutical compositions disclosed herein.

As used herein, the term "sensitization", "sensitization" or "sensitizer" refers to an increase in sensitivity or a decrease in resistance of a cancer sample or mammal in response to a treatment. According to methods known In the art and described below for specific treatments, including, for example, cell proliferation assays (Tanigawa N, Kem DH, Kikasa Y, Morton DL, Cancer Res, 42: 2159-, mice were 4-6 weeks old. If the increase in treatment sensitivity or decrease in resistance is 25% or more, for example 30%, 40%, 50%, 60%, 70%, 80% or up to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more compared to treatment sensitivity or resistance in the absence of such compositions or methods. Determination of sensitivity or resistance to therapeutic treatment is routine in the art and within the skill of the ordinarily skilled clinician.

As used herein, "cancer therapy sensitizer" refers to a compound or composition comprising at least one compound that can sensitize a cancer therapy. For example, it refers to a compound or composition comprising an effective amount of at least one compound and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof. For example, the above-described compounds or compositions can be administered prior to, during, or prior to cancer treatment to ameliorate or enhance the effect of one or more therapeutically active compositions on a cancer or tumor in an individual in need thereof, and then achieve the goal of eliminating, inhibiting, ameliorating, comforting, or preventing the cancer and its symptoms; the speed of tumor proliferation is delayed, prohibited and reversed; or a medical effect similar to the above purpose.

As used herein, the term "chemotherapy" refers to the use of chemical agents to destroy cancer cells. Exemplary chemotherapeutic agents include, but are not limited to, actinomycin D, doxorubicin, adriamine, asparaginase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, CPT-11, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, epirubicin etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, phosphoramide, irinotecan, doxorubicin liposomes, lomustine, melphalan, mercaptopurine, methotrexate, mitomycin, mitoxantrone, oxaliplatin, procarbazine, thiotrimethoprim of paclitaxel, methylthiomethylthiomethylthiomethylthiomethylthiomethylthiomethoprim, thiomethoxazole, topotecan, treosulfan, UFT (Uracil-Tegufur), vinblastine, vincristine, vindesine, and vinorelbine. Chemotherapy may be used alone or in combination to treat certain types of cancer. Sometimes it can be used with other types of treatment, such as surgery, radiation therapy, immunotherapy, or a combination thereof.

As used herein, "radiotherapy," also referred to as "radiotherapy," refers to the treatment of cancer and other diseases with ionizing radiation. Ionizing radiation deposits energy that damages or destroys cells in the area being treated (i.e., the "target tissue") by destroying the genetic material of the cells, rendering them unable to continue growing. While radiation can damage cancer cells and normal cells, normal cells are capable of self-repair and function normally. Radiotherapy may be used to treat a local solid tumour, such as skin cancer, tongue cancer, larynx cancer, brain cancer, breast cancer or cervical cancer. It can also be used for the treatment of leukemia and lymphoma (cancers of the hematopoietic and lymphatic systems, respectively). Exemplary radiation therapy may be selected from the form of electromagnetic waves, such as X-rays or gamma rays, or charged or neutral particles. Radiation therapy may be delivered by external irradiation, interstitial implants, or a combination thereof. The majority of tumors were treated for 60-70G in 4-7 weeks_yThe course of radiotherapy.

The radiation and chemotherapy sensitizers disclosed herein are pharmaceutical compounds or compositions that can be used prior to or simultaneously with radiation and chemotherapy to enhance the effect of radiation and chemotherapy on tumors.

As used herein, the term "administering" or "administering" refers to introducing a compound or composition (e.g., a therapeutic agent) into the body of a mammal by any means to prevent or treat a disease or disorder (e.g., cancer).

As used herein, the terms "treatment," "therapy," and "therapeutic treatment" as used herein refer to either curative therapy or prophylactic therapy. These terms also describe the management and care of a mammal for combating a disease or related condition, and include the administration of compositions to alleviate symptoms, side effects, or other complications of a disease or condition. Treatment of cancer includes, for example, surgery, chemotherapy, radiation therapy, gene therapy, and immunotherapy.

As used herein, the term "mammal" refers to a human or other animal, such as a farm animal or a laboratory animal (e.g., guinea pig or mouse). In some embodiments, the mammal is a human. May be a human who has been diagnosed as in need of treatment for a disease or condition disclosed herein.

"pharmaceutically effective amount" includes an amount sufficient to ameliorate or prevent a symptom or sign of a medical condition. The effective amount for a particular patient or veterinary subject can vary depending on a variety of factors, such as the condition being treated, the overall health of the patient, the method and dosage of administration, and the severity of the side effects. The pharmaceutically effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. The effect will result in an improvement of the diagnostic measure or parameter of at least 5%, such as at least 10%, further such as at least 20%, further such as at least 30%, further such as at least 40%, further such as at least 50%, further such as at least 60%, further such as at least 70%, further such as at least 80%, even further such as at least 90%, wherein 100% is defined as the diagnostic parameter exhibited by normal subjects. A pharmaceutically effective amount of crocin is, for example, an amount sufficient to reduce tumor volume, inhibit tumor growth, or prevent or reduce metastasis before, during, or after cancer treatment.

The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, and suitable for use in contact with human or animal tissue.

The pharmaceutical compositions disclosed herein may be a conventional tablet base such as lactose, sucrose and corn starch with a binder such as acacia, corn starch or gelatin, a disintegrant such as potato starch, alginic acid, corn starch and guar gum, tragacanth, acacia, which is intended to aid disintegration and dissolution of the tablet upon administration, a lubricant such as talc, stearic acid, or magnesium, calcium or zinc stearate, dyes, colorants and flavoring agents (such as peppermint, oil of wintergreen or cherry flavoring) which are intended to improve the tablet granulation flow and prevent sticking of tablet materials to the tablet die and punch surfaces, and to enhance the aesthetic qualities of the tablet and make them more acceptable to patients.

Suitable excipients for oral liquid dosage forms include, for example, dicalcium phosphate and diluents such as water and alcohols, for example ethanol, benzyl alcohol and polyvinyl alcohol, with or without the addition of pharmaceutically acceptable surfactants, suspending agents or emulsifying agents. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar or both. Dispersible powders and granules are suitable for the preparation of aqueous suspensions. They provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Examples of suitable dispersing or wetting agents and suspending agents are those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, such as those described above, may also be present.

The pharmaceutical compositions disclosed herein may also be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil, for example liquid paraffin or a mixture of vegetable oils. Suitable emulsifying agents may, for example, be selected from (1) naturally-occurring gums, for example gum acacia and gum tragacanth, (2) naturally-occurring phosphatides, for example soy bean and lecithin, (3) esters or partial esters of acids derived from fats and hexitol anhydrides, for example sorbitan monooleate, (4) condensation products of the partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents, for example.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. The suspension may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate; one or more colorants; one or more flavoring agents; and one or more sweetening agents, such as sucrose or saccharin.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also comprise, for example, at least one entity chosen from demulcents, preservatives such as methyl and propyl parabens, and flavoring and coloring agents.

The combinations disclosed herein can also be administered parenterally, i.e., subcutaneously, intravenously, intraocularly, intramuscularly or intraperitoneally, as injectable doses of crocetin, for example in a physiologically acceptable diluent, the pharmaceutically acceptable carrier can be a sterile liquid or a mixture of liquids, such as water, saline, aqueous dextrose and related sugar solutions, alcohols (e.g., ethanol, isopropanol or cetyl alcohol), glycols (e.g., propylene glycol or polyethylene glycol), glycerol ketals (e.g., 2-dimethyl-1, 1) -dioxolane-4-methanol, ethers such as polyethylene glycol) 400, oils, fatty acids, fatty acid esters or fatty acid glycerides or acetylated fatty acid glycerides, with or without the addition of pharmaceutically acceptable surfactants such as soaps or detergents, suspending agents such as pectin, carbomer, methylcellulose, hydroxypropylmethylcellulose or carboxymethylcellulose, or emulsifiers and other pharmaceutical adjuvants.

Examples of oils useful in the parenteral formulations disclosed herein are those of petroleum, animal, vegetable or synthetic origin, selected from, for example, peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum and mineral oil. Suitable fatty acids include oleic acid, stearic acid, isostearic acid and myristic acid. Suitable fatty acid esters are, for example, ethyl oleate and isopropyl myristate. Suitable soaps include fatty acid alkali metal, ammonium and triethanolamine salts, and suitable detergents include cationic detergents such as dimethyl dialkyl ammonium halides, alkyl pyridinium halides and alkylamine acetates; anionic detergents such as alkyl, aryl and olefin sulfonates, alkyl, olefin, ether and monoglyceride sulfates, and sulfosuccinates; nonionic detergents such as fatty amine oxides, fatty acid alkanolamides, and poly (oxyethylene-oxypropylene) or ethylene oxide or propylene oxide copolymers; and amphoteric detergents such as alkyl-beta-aminopropionates and 2-alkylimidazoline quaternary ammonium salts, and mixtures thereof.

Examples of surfactants for use in parenteral formulations are polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol.

The pharmaceutical compositions disclosed herein may be in the form of a sterile injectable aqueous suspension. Such suspensions may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents, for example, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinyl pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be naturally occurring phosphatides, for example lecithin, condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadeca-ethoxyhexadecanol, condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate.

The sterile injectable preparation disclosed herein may also be a sterile injectable solution or suspension in, for example, a non-toxic parenterally-acceptable diluent or solvent. Diluents and solvents which may be used are, for example, water, ringer's solution, isotonic sodium chloride solution and isotonic glucose solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The pharmaceutical compositions disclosed herein may also be administered in the form of suppositories for rectal administration of the drug. For example, these compositions may be prepared by mixing the drug with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. These materials are selected, for example, from cocoa butter and polyethylene glycols.

Controlled release formulations for parenteral administration include, for example, liposome, polymeric microsphere and polymeric gel formulations known in the art.

It may be desirable or necessary to introduce a pharmaceutical composition disclosed herein into a patient via a mechanical delivery device. The construction and use of mechanical delivery devices for delivering pharmaceutical agents is well known in the art. For example, direct techniques for direct drug delivery to the brain typically involve placing a drug delivery catheter into the ventricular system of a patient to bypass the blood brain barrier. One such implantable delivery system is described in U.S. patent No.5,011,472 for delivering an agent to a specific anatomical region of the body.

The pharmaceutical compositions disclosed herein may also contain other conventional pharmaceutically acceptable ingredients, commonly referred to as carriers, diluents, or adjuvants, as needed or desired. Conventional methods of preparing such compositions in suitable dosage forms may be used. These ingredients and procedures include those described in the following references: powell, M.F., et al, "summary of excipients for parenteral preparations", PDA Journal of Pharmaceutical Science ft Technology 52(5), 238-311 (1998); strickley, R.G "parenteral formulation of small molecule therapeutic drugs marketed in the united states (1999) -part 1" PDA Journal of Pharmaceutical Science & Technology 53(6), "324-349 (1999); and Nema, S. Et al, "excipients and their use in injectable products," PDA Journal of Pharmaceutical Science Et Technology, 51(4), 166- "171 (1997). Common pharmaceutical ingredients that may be used as appropriate to formulate the compositions disclosed herein for their intended route of administration include, for example, acidulants (examples include, but are not limited to, acetic acid, citric acid, fumaric acid, hydrochloric acid, and nitric acid); alkalizing agents (examples include, but are not limited to, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium hydroxide and triethanolamine, triethanolamine); adsorbents (examples include, but are not limited to, powdered cellulose and activated carbon); aerosol propellants (examples include, but are not limited to, carbon dioxide, chlorofluorocarbons such as Freon-11(CCl3F), Freon-13(CClF3), and Freon-114 (primarily CCIF2-CCIF 2)); air displacement agents (examples include, but are not limited to, nitrogen and argon); antifungal preservatives (examples include, but are not limited to, benzoic acid, butyl paraben, ethyl paraben, methyl paraben, propyl paraben, and sodium benzoate); antimicrobial preservatives (examples include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, and thimerosal); antioxidants (examples include, but are not limited to, ascorbic acid, ascorbyl palmitate, butyl hydroxyanisole, butyl hydroxytoluene, hypophosphorous acid, thioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, and sodium metabisulfite); adhesive materials (examples include, but are not limited to, block polymers, natural and synthetic rubbers, polyacrylates, polyurethanes, silicones, polysiloxanes, and styrene-butadiene copolymers); buffering agents (examples include, but are not limited to, potassium metaphosphate, dipotassium phosphate, sodium acetate, sodium citrate anhydrous, and sodium citrate dihydrate); a carrier (such as, but not limited to, acacia syrup, aromatic elixir, cherry syrup, cocoa syrup, orange juice, syrup, corn oil, mineral oil, peanut oil, sesame oil, bacteriostatic sodium chloride for injection, and bacteriostatic water for injection); chelating agents (examples include, but are not limited to, edetate disodium and edetic acid); coloring agents (examples include, but are not limited to FD & C Red No.3, FD & C Red No.20, FD & C Yellow No.6, FD & C Blue No.2, D & C Green No.5, D & C Orange No.5, D & C Red No.8, caramel, and Red iron oxide); clarifying agents (examples include, but are not limited to, bentonite); emulsifying agents (examples include, but are not limited to, acacia, cetomolegol, cetyl alcohol, glyceryl monostearate, lecithin, sorbitan monooleate, and polyoxyethylene 50 monostearate); encapsulating agents (examples include, but are not limited to, gelatin and cellulose acetate phthalate); flavorants (examples include, but are not limited to, anise oil, cinnamon oil, cocoa, menthol, orange oil, peppermint oil, and vanillin); humectants (examples include, but are not limited to, glycerin, propylene glycol, and sorbitol); planktonic agents (examples include, but are not limited to, mineral oil and glycerin); oils (examples include, but are not limited to, peanut oil, mineral oil, olive oil, peanut oil, sesame oil, and vegetable oil); ointment bases (examples include, but are not limited to, lanolin, hydrophilic ointment, polyethylene glycol ointment, petrolatum, hydrophilic petrolatum, white ointment, yellow ointment, and rose water ointment); penetration enhancers for e.g. transdermal delivery (examples include, but are not limited to, monohydric or polyhydric alcohols, saturated or unsaturated fatty acid esters, saturated or unsaturated dicarboxylic acids, essential oils, phosphatidyl derivatives, cephalins, terpenes, amides, ethers, ketones, and ureas)

Plasticizers (examples include, but are not limited to, diethyl phthalate and glycerol); solvents (examples include, but are not limited to, ethanol, corn oil, cottonseed oil, glycerol, isopropanol, mineral oil, oleic acid, peanut oil, purified water, and sterile water for injection; hardeners (examples include, but are not limited to, cetyl alcohol, cetyl esters wax, microcrystalline wax, paraffin, stearyl alcohol, white wax, and yellow wax); suppository bases (examples include, but are not limited to, cocoa butter and polyethylene glycols); surfactants (examples include, but are not limited to, benzalkonium chloride, nonoxynol 10, oxybenzene alcohol 9, polysorbate 80, sodium lauryl sulfate, and sorbitan monopalmitate); suspending agents (examples include, but are not limited to, agar, bentonite, carbomer, sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, kaolin, methylcellulose, tragacanth and veegum); sweetening agents (examples include, but are not limited to, aspartame, dextrose, glycerin, mannitol, propylene glycol, sodium saccharin, sorbitol, and sucrose); tablet antiadherents (examples include, but are not limited to, magnesium stearate and talc); tablet binders (examples include, but are not limited to, acacia, alginic acid, sodium carboxymethylcellulose, compressible sugar, ethylcellulose, gelatin, liquid glucose, methylcellulose, non-crosslinked polyvinylpyrrolidone, and pregelatinized starch); tablet and capsule diluents (examples include, but are not limited to, dibasic calcium phosphate, kaolin, lactose, mannitol, microcrystalline cellulose, powdered cellulose, precipitated calcium carbonate, sodium phosphate, sorbitol, and starch); tablet coatings (examples include, but are not limited to, liquid glucose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, ethylcellulose, cellulose acetate phthalate, and shellac); tablet direct compression excipients (examples include, but are not limited to, dibasic calcium phosphate); tablet disintegrating agents (examples include, but are not limited to, alginic acid, carboxymethylcellulose calcium, microcrystalline cellulose, polacrilin potassium, cross-linked polyvinylpyrrolidone, sodium alginate, sodium starch glycolate, and starch); tablet glidants (examples include, but are not limited to, colloidal silicon dioxide, corn starch, and talc); tablet lubricants (examples include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, stearic acid, and zinc stearate); tablet/capsule opacifiers (examples include, but are not limited to, titanium dioxide); tablet polishes (examples include, but are not limited to, carnauba wax and white wax); thickening agents (examples include, but are not limited to, beeswax, cetyl alcohol, and paraffin wax); tonicity agents (examples include, but are not limited to, dextrose and sodium chloride); viscosity increasing agents (examples include, but are not limited to, alginic acid, bentonite, carbomer, sodium carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone, sodium alginate, and tragacanth); and wetting agents (examples include, but are not limited to, heptadecaethylene oxycetanol, lecithin, sorbitol monooleate, polyoxyethylene sorbitol monooleate, and polyoxyethylene stearate).

When the sensitiser liquid or gel disclosed herein is in the form of an injection, the injection may be directly intratumorally injected using, for example, a syringe, or indirectly injected into the tumor region upon irradiation, for example, via an angiographic catheter. In such cases, the sensitizer liquid or gel disclosed herein may be injected into the targeted tumor area using, for example, a syringe or angiographic catheter, before, after, or concurrently with administration of the anti-cancer agent. For example, intratumoral injection can be performed under ultrasound examination guidance using a syringe with an approximately 21 gauge needle while observing the status of penetration of the sensitizer into the tissue. The sensitizer may be delivered extensively to the tissue under ultrasound guidance to alter the depth and direction of the injection needle. The dose of sensitizer administered to the tumor area will vary depending on the size of the tumor and the method of administration.

Anticancer agents for use herein include, for example, alkylating agents such as cyclophosphamide, ifosfamide, busulfan, melphalan, bendamustine hydrochloride, nimustine hydrochloride, ranimustine, dacarbazine, procarbazine hydrochloride, and temozolomide; antimetabolites such as methotrexate, pemetrexed sodium, fluorouracil, doxifluridine, capecitabine, tegafur, cytarabine octaphosphate hydrate, decitabine, gemcitabine hydrochloride, mercaptopurine hydrate, fludarabine phosphate, potassium levo-glycosaminoglycan, calcium levo-glycosaminoglycan, and azacitidine; antitumor antibiotics, such as doxorubicin hydrochloride, daunorubicin hydrochloride, pirarubicin, epirubicin hydrochloride, idarubicin hydrochloride, aclarubicin hydrochloride, amrubicin hydrochloride, mitoxantrone hydrochloride, mitomycin C, actinomycin D, bleomycin, pemetrexed sulfate, zoledrycin; microtubule inhibitors such as vincristine sulfate, vinblastine sulfate, vindesine sulfate, vinorelbine tartrate, paclitaxel, docetaxel hydrate, and eribulin mesylate; hormonal agents such as anastrozole, exemestane, letrozole, tamoxifen citrate, toremifene citrate, fulvestrant, flutamide, bicalutamide, medroxyprogesterone acetate, estramustine sodium phosphate hydrate, and leuprorelin acetate; platinum drugs such as cisplatin, miriplatin hydrate, carboplatin, nedaplatin, oxaliplatin; topoisomerase I inhibitors such as irinotecan hydrochloride hydrate and norgetecan hydrochloride; topoisomerase II inhibitors, such as etoposide and sobuzole; cytokines such as interferon γ 1a, teceleukin, and celloleukin; antibody drugs such as trastuzumab, rituximab, gemtuzumab ozolomide, bevacizumab, and cetuximab; radioimmunotherapeutic agents, such as ibritumomab tiuxetan; molecularly targeted drugs such as gefitinib, imatinib mesylate, bortezomib, erlotinib hydrochloride, sorafenib tosylate, sunitinib malate, thalidomide, nilotinib hydrochloride hydrate, dasatinib hydrate, lapatinib tosylate hydrate, everolimus, lenalidomide hydrate, deslimitamine, and non-specific immunostimulants such as OK-432, dry bacillus calmette-guerin, coriolus versicolor polysaccharide preparations, lentinan and ubenimex. Other examples of anticancer agents include acetogelator, sodium porphyrin, talaporfin sodium, ethanol and arsenic trioxide.

Examples of the anticancer agent include anthracycline anticancer agents such as doxorubicin hydrochloride, daunorubicin hydrochloride, pirarubicin, epirubicin hydrochloride, idarubicin hydrochloride, aclarubicin hydrochloride, amrubicin hydrochloride, and mitoxantrone hydrochloride; platinum anticancer agents such as cisplatin, miriplatin hydrate, carboplatin, nedaplatin, and oxaliplatin; pyrimidine antimetabolite based anticancer agents such as fluorouracil, doxifluridine, capecitabine, tegafur, cytarabine octaphosphate hydrate, escitalopram and gemcitabine hydrochloride.

The present disclosure, by way of the following examples, has demonstrated that crocin can be successfully used, for example, as a chemotherapy and/or radiotherapy sensitizer for various solid tumors transplanted onto mice. It can act synergistically with radiotherapy and/or chemotherapy to prolong the survival of tumor-bearing mice. Furthermore, crocin may have the following advantages compared to TSC currently in phase III clinical trials: (1) the overall survival of tumor-bearing mice treated with crocin in combination with radiotherapy and chemotherapy can be significantly longer than those treated with the same dose of TSC in combination with radiotherapy and chemotherapy; (2) crocin can not only enhance the sensitivity of tumor cells to radiotherapy, but also enhance the sensitivity of tumor cells to chemotherapy; (3) the optimal dose for maximum synergy of TSC with radiotherapy and chemotherapy is 100 μ g/kg, followed by a dose-dependent decrease, while the synergy of crocin with radiotherapy and chemotherapy may be a dose-dependent increase. The range is 100-400. mu.g/kg.

The scope of the disclosure may be better understood with reference to the following examples, which are not intended to limit the disclosure to the particular examples.

Example 1 preparation of crocetin drug-loaded liposome formulations

13mg of lecithin, 5mg of cholesterol, and 2mg of distearoyl phosphatidyl ethanolamine-polyethylene glycol (PEG-DSPE2000) were weighed, dissolved in 2ml of chloroform, 3mg of the drug (dissolved in DMSO) was added, and the mixture was placed in a film-forming eggplant-shaped flask and subjected to vacuum recovery of a chloroform organic solvent to form a film. Adding 2ml of deionized water into the eggplant-shaped flask, and rotationally hydrating to obtain the drug-loaded liposome.

Example 2 inhibition of the BALB/c nude mouse solid tumor model by combination of crocetin with radiotherapy

1. Experimental Material

Hela human cervical cancer cell line, HCT116 human colon cancer cell line, HepG2 human liver cancer cell line, (ATCC).

Crocetin: brick red powder, purity greater than 98% by HPLC (shaoxingtang biotechnology limited).

The preparation method comprises the steps of preparing the liposome preparation prepared in example 1 into crocetin for intravenous injection, and preparing crocetin suspension with a certain concentration by 0.5% sodium carboxymethylcellulose for intragastric administration.

TSC: according to the patent method (US 606060511), the crocetin is used for preparation, and the purity is more than 98 percent by HPLC detection.

Male BALB/c nude mice, 4-8 weeks old, were supplied by the animal center of the institute of medical sciences, Zhejiang province.

2. Method of producing a composite material

2.1 Molding

Collecting well-grown Hela, HCT116 and HepG2 human tumor cell lines, and suspending the cell lines into 1x10 concentration by PBS⁷Suspension of/m 1, 200ul (2X 10) of subcutaneous right forelimb axilla per BALB/c nude mouse (4-6 weeks old)⁶) The inoculation is carried out by ensuring that the inoculation part of each mouse and the number of inoculated tumor cells are consistent, 4 cells are inoculated in each cell strain, and 12 cells are inoculated in total. When the tumor volume is increased to 0.3 cubic centimeter, tumor tissues which grow well, have no degeneration necrosis, are light red and fish-shaped are selected for inoculation, tumor blocks with the diameter of about 2mm are inoculated, and 25 tumors are inoculated in each tumor tissue.

2.2 dosing intervention

Inoculating and growing for 2-3 weeks, after the volume of tumor mass reaches 0.25-0.35 cubic centimeter, randomly dividing 25 mice in each group into 5 groups,

(1) model Control (Control);

(2) radiotherapy group (RT);

(3) radiotherapy + intravenous TSC group (RT + TSC iv (100 ug/kg));

(4) radiotherapy + intravenous Crocetin group (RT + Crocetin iv (100 ug/kg));

(5) radiotherapy + oral Crocetin group (RT + Crocetin po (200 ug/kg)).

All mice were dosed for 5 consecutive days, and study groups 2-5 were subjected to a single, local 5Gy of XRay radiation (RS-2000-PRO-225(RAD SOURCE)) for 30 minutes after dosing on day 3.

2.3 Experimental observations

Tumor volumes were measured 2-3 times per week for all study groups until tumor volumes reached 4 times the volume at the start of treatment. Tumor volumes stored for each group of mice were calculated: tumor volume is 0.5 × length × width²(ii) a Relative tumor volume (Vt) measured on day/tumor volume on day one (V0) x 100% on day one.

2.4 statistical methods

The data analysis adopts a statistical program software package (SPSS 21.0 for Windows) to carry out statistical analysis, a relative tumor volume-time curve is drawn, the statistical difference between different test groups is analyzed by using single-factor variance, and the difference is significant when P is less than 0.05.

3. Results

Referring to figures 1 to 3, wherein figure 1 shows the inhibitory effect of crocetin or TSC in combination with radiotherapy on Hela subcutaneous tumors; figure 2 shows the inhibitory effect of crocetin or TSC in combination with radiotherapy on HCT116 subcutaneous tumors and figure 3 shows the inhibitory effect of crocetin or TSC in combination with radiotherapy on HepG2 subcutaneous tumors. Compared with the control group, other treatment groups can effectively inhibit the subcutaneous tumor enlargement (p is less than 0.05) after the administration or the radiotherapy is finished (after 5 d); compared with the RT group, the treatment effect of the RT combined with TSC or crocetin is improved to different degrees (P is less than 0.01) after 6 days; compared with the TSC + RT group, the treatment effect (after 6 d) of the RT combined with the crocetin (intragastric or intravenous injection) is better than that of the RT combined with the TSC (P is less than 0.01), and in conclusion, the inhibition effect of the crocetin combined with radiotherapy on a human tumor in vitro model has the statistically significant advantage (P is less than 0.05) compared with other treatment groups.

4. Conclusion

The tests prove that the crocetin can obviously enhance the inhibition effect of radiotherapy on the growth of various solid tumors and delay the growth of subcutaneous tumors.

Example 3 inhibitory Effect of crocetin in combination with temozolomide on BALB/C nude mouse C6 orthotopic glioma model

1. Experimental Material

C6 glioma cell line (ATCC).

Temozolomide, more than 99% pure (guan-shou life science ltd, guang).

2. Method of producing a composite material

2.1 Molding

The well-grown C6 glioma cell line was suspended with PBS to a concentration of 2X106/10ul, and each mouse was inoculated with 2.5ul (5X 10) intracranial⁵Individual cells), which were inoculated to ensure consistent numbers of tumor cells and inoculated sites per mouse, for a total of 50.

2.2 dosing intervention

7 days after inoculation, 50 mice were randomly divided into 6 groups of 8-9 mice each;

(1) model Control (Control);

(2) temozolomide group (TMZ); (ii) a

(3) Temozolomide + intravenous TSC group (TMZ + TSC (100 ug/kg));

(4) temozolomide + group of intravenous Crocetin (TMZ + Crocetin (100 ug/kg));

(5) set of TSC (100 ug/kg));

(6) crocetin group (Crocetin (100ug/kg)) was administered intravenously.

All mice were injected intravenously with TSC, crocetin or saline (control) from 10-14 days for 5 consecutive days, and after 20 minutes, replaced with mochiThe zolamide is administered by intraperitoneal injection (50mg/kg, 149 mg/m)²)。

2.3 Experimental observations

Observing the autonomous activity, mental state, hair, respiration, diet and fecal characters of the mice and the response to external stimulation every day, continuously weighing the body weight of each group of mice in the survival period, recording the survival period of each group of mice in all administration periods, and calculating the survival rate of each group of mice: survival rate (number of remaining animals per group/total number of animals per group).

2.4 statistical methods

The data analysis adopts a statistical program software package (SPSS 21.0 for Windows) to carry out statistical analysis, a Kaplan-Meier method is used for drawing a survival curve, a Log Rank test (Log Rank (Mantel-Cox)) is used for determining the difference between survival distributions of different test groups, and the difference is significant if P is less than 0.05.

3. Results

FIG. 4 shows the survival time of the groups of mice as a function of the number of days observed and the statistical variance between the survival curves of the groups of mice; the temozolomide can obviously prolong the survival period of nude mice with in-situ planted tumors, the crocetin can further prolong the survival period of the temozolomide to the nude mice with in-situ planted tumors (P is less than 0.01), and the TSC has no prolonging effect. The survival period of nude mice with in-situ planted tumors cannot be prolonged by single intravenous injection of TSC or crocetin.

4. Conclusion

The tests prove that the crocetin can obviously enhance the inhibition effect of temozolomide on a BALB/C nude mouse C6 in-situ glioma model and prolong the life cycle of in-situ planted tumor nude mice.

Example 4 inhibition of BALB/C nude mouse C6 orthotopic glioma model by combination of crocetin with radiotherapy and temozolomide

1. Experimental Material

C6 glioma cell line (ATCC).

Temozolomide, more than 99% pure (guan-shou life science ltd, guang).

2. Method of producing a composite material

2.1 Molding

Taking well-grown C6 glioma cell strain, and suspending the cell strain into PBS with the concentration of 2x10⁶10ul suspension, 2.5ul (5X 10) intracranial inoculation per mouse⁵) And the inoculation is carried out on the same number of inoculated parts and inoculated tumor cells of each mouse, and 50 mice are inoculated in total.

2.2 dosing intervention

(1) model Control (Control);

(2) temozolomide + radiotherapy group (TMZ + RT);

(3) temozolomide + radiotherapy + intravenous TSC group (TMZ + RT + TSC (100 ug/kg));

(4) temozolomide + radiotherapy + intravenous crocetin group (TMZ + RT + TK10iv (100 ug/kg));

(5) temozolomide + radiotherapy + intravenous crocetin group (TMZ + RT + TK10iv (200 ug/kg));

(6) temozolomide + radiotherapy + oral crocetin group (TMZ + RT + TK10po (200 ug/kg).

All mice were administered either TSC, crocetin or saline (control) intravenously for 5 consecutive days from 7-11 days, and temozolomide was administered intraperitoneally 20 minutes after administration (50mg/kg, 149 mg/m)²). Within 30 minutes after dosing on day 5, mice from groups 2-6 received 8Gy irradiation (RS-2000-PRO-225(RAD SOURCE)).

(3) Experimental observation

(4) Statistical method

3. Results

FIG. 5 shows the survival time of the groups of mice as a function of the number of days observed and the statistical variance between the survival curves of the groups of mice; the survival period of nude mice with in-situ planted tumors can be obviously prolonged by temozolomide and radiotherapy, and the survival period of nude mice with in-situ planted tumors can be further obviously prolonged by the temozolomide and radiotherapy (P is less than 0.01).

4. Conclusion

The tests prove that the crocetin can enhance the inhibition effect of radiotherapy and temozolomide on a BALB/C nude mouse C6 in-situ glioma model and prolong the life cycle of nude mice implanted with tumors in situ.

Example 5 inhibition of the BALB/C nude mouse C6 orthotopic glioma model by combination of crocetin with radiotherapy and temozolomide for two consecutive courses of treatment

1. Experimental Material

C6 glioma cell line (ATCC).

Temozolomide, more than 99% pure (guan-shou life science ltd, guang).

2. Method of producing a composite material

2.1 Molding

Taking well-grown C6 glioma cell strain, and suspending the cell strain into PBS with the concentration of 2x10⁶10ul suspension, 2.5ul (5X 10) intracranial inoculation per mouse⁵) And the inoculation time ensures that the inoculation part of each mouse and the number of inoculated tumor cells are consistent.

2.2 dosing intervention

6 days after inoculation, 41 mice were randomly divided into 5 groups of 8-9 mice each;

(1) model Control group (Control)

(2) Temozolomide + radiotherapy group (TMZ + RT);

(5) temozolomide + radiotherapy + oral crocetin group (TMZ + RT + TK10po (200 ug/kg)).

First-stage administration: all mice were administered intravenous TSC, crocetin or normal saline (control) for 5 consecutive days from 7-11 days, and after 20 minutes, temozolomide was administered intraperitoneally (50mg/kg, 149 mg/m)²). Within 30 minutes after the 5 th day dosing, mice from groups 2-6 received topical 8Gy irradiation (RS-2000-PRO-225(RAD SOURCE)).

And (3) second-stage administration: all mice were administered intravenous TSC, crocetin or normal saline (control) for 5 consecutive days from 16-20 days, and after 20 minutes, temozolomide was administered intraperitoneally (50mg/kg, 149 mg/m)²)。

2.3 Experimental observations

2.4 statistical methods

3. Results

FIG. 6 shows the survival time of each group of mice as a function of the number of days observed and the statistical variance between the survival curves of each group of mice; the survival period of nude mice with in-situ planted tumors can be obviously prolonged by temozolomide and radiotherapy, the survival period of nude mice with in-situ planted tumors can be further obviously prolonged by TSC and crocetin in two continuous treatment courses (P is less than 0.01), and the improvement effect of crocetin is more obvious than that of TSC (P is less than 0.05).

4. Conclusion

The tests prove that the crocetin can obviously enhance the inhibition effect of radiotherapy and temozolomide on a BALB/C nude mouse C6 in-situ glioma model and prolong the survival period of nude mice implanted with tumors in situ.

Example 6 inhibition of C57BL/6 mouse homolog GL261 in situ glioma model by combination of crocetin with radiation therapy and temozolomide

1. Experimental Material

GL261 glioma cell line (ATCC).

Temozolomide, more than 99% pure (guan-shou life science ltd, guang).

Male C57BL/6 mice, 4-8 weeks old, were obtained from the animal center of the institute of medical sciences, Zhejiang province.

2. Method of producing a composite material

2.1 Molding

Collecting well-grown GL261 glioma cell line, and suspending with PBS to concentration of 1 × 10⁶10ul suspension, 2ul (2X 10) intracranial inoculation per mouse⁵) When in inoculation, the inoculation part of each mouse and the number of inoculated tumor cells are ensured to be consistent, and 49 mice are inoculated in total.

2.2 dosing intervention

After 9 days of inoculation, 49 mice were randomly divided into 7 groups of 5-8 mice each;

(1) model Control (Control);

(2) temozolomide + radiotherapy group (TMZ + RT);

(5) temozolomide + radiotherapy + intravenous crocetin group (TMZ + RT + TK10iv (300 ug/kg));

(6) temozolomide + radiotherapy + oral crocetin group (TMZ + RT + TK10po (200 ug/kg)).

All mice were dosed from 9-11 days, 3 consecutive days: intravenous injection of TSC, crocetin or normal saline on day 9 (control); intravenous injection of TSC, crocetin or normal saline is performed on day 10, and after 20 minutes, intraperitoneal injection of temozolomide is performed (100mg/kg, 298.5 mg/m)²) (ii) a Mice of groups 2-7 received topical 5Gy irradiation (RS-2000-PRO-225(RAD SOURCE)) within 30 minutes after the first intravenous injection of TSC, crocetin or saline on day 11.

2.3 Experimental observations

2.4 statistical methods

3. Results

FIG. 7 shows the Kaplan-Meier survival curves for each group of mice and the statistical parameters between the survival curves for each group of mice; compared with the control group, the survival period of the mice with the glioma in situ can be effectively prolonged by other treatment groups (P is less than 0.05), and the median survival time is prolonged from 25 days to more than 36 days; compared with the TMZ + RT group, the treatment effect of the TMZ + RT is improved to different degrees (P is less than 0.05) after the combination of the TSC or the crocetin, and the median survival rate is improved from 36 days to more than 45 days; compared with the TSC + TMZ + RT group, the treatment effect of the combination of the Crocetin (intragastric or intravenous injection) is better than that of the combination of the TSC (P is less than 0.05), the median survival time is prolonged from 45 days to more than 47 days, and in conclusion, the animals treated by the combination of the Crocetin + TMZ + RT have statistically significant survival advantage (P is less than 0.05) compared with other treatment groups.

4. Conclusion

The tests prove that the crocetin can obviously enhance the inhibition effect of radiotherapy and temozolomide on a GL261 in-situ glioma model of a C57BL/6 mice, and prolong the survival period of nude mice with in-situ planted tumors.

Example 7 dose-dependent inhibition of C57BL/6 mouse homologous GL261 in situ glioma model by combination of crocetin with radiotherapy and temozolomide

1. Experimental Material

GL261 glioma cell line (ATCC).

Temozolomide, more than 99% pure (guan-shou life science ltd, guang).

2. Method of producing a composite material

2.1 Molding

Collecting well-grown GL261 glioma cell line, and suspending with PBS to concentration of 1 × 10⁶10ul suspension, 2ul (2X 10) intracranial inoculation per mouse⁵) And the inoculation time ensures that the inoculation part of each mouse and the number of inoculated tumor cells are consistent.

2.2 dosing intervention

11 days after inoculation, 42 mice were randomly divided into 5 groups of 8-9 mice each;

(1) model Control (Control);

(2) temozolomide + radiotherapy group (TMZ + RT);

(3) temozolomide + radiotherapy + intravenous Crocetin group (TMZ + RT + Crocetin iv (100 ug/kg));

(4) temozolomide + radiotherapy + intravenous Crocetin group (TMZ + RT + Crocetin iv (200 ug/kg));

(5) temozolomide + radiotherapy + intravenous Crocetin group (TMZ + RT + Crocetin iv (400 ug/kg));

all mice were dosed from 11-13 days, 3 consecutive days: intravenous crocetin or normal saline on day 11 (control); on day 12, crocetin or normal saline is injected intravenously, and after 20 minutes, temozolomide is injected intraperitoneally (100mg/kg, 298.5 mg/m)²) (ii) a Mice in groups 2-5 received topical 5Gy irradiation (RS-2000-PRO-225(RAD SOURCE)) within 30 minutes after the first intravenous injection of crocetin or saline on day 13.

2.3 Experimental observations

2.4 statistical methods

3. Results

FIG. 8 shows the Kaplan-Meier survival curves for each group of mice and the statistical parameters between the survival curves for each group of mice; compared with the control group, the survival period of the mice with the glioma in situ can be effectively prolonged by other treatment groups (P is less than 0.01), and the median survival time is prolonged from 24 days to more than 35 days; compared with the TMZ + RT group, after the combination of the crocetin, the treatment effects of the TMZ + RT are improved to different degrees (P is less than 0.01), and the median survival rate is improved from 35 days to more than 44 days; and 3 groups of crocetin with different concentrations have obvious difference (P is less than 0.05) between the TMZ + RT groups, which is represented by that the higher the crocetin dosage is, the longer the survival median is, and the days are from 44 to 49; in conclusion, animals treated with the combination of Crocetin + TMZ + RT had a statistically significant survival advantage (P < 0.01) compared to the other treatment groups, and Crocetin was able to dose-dependently prolong the survival of animals treated with the combination of TMZ + RT.

4. Conclusion

Claims

1. A method of treating a solid tumor in a mammal comprising administering a pharmaceutically effective amount of crocetin to the mammal prior to, during, or after treatment with radiation therapy, chemotherapy, immunotherapy, or a combination thereof.

2. The method of claim 1, wherein the mammal is a human.

3. The method of claim 1, wherein the radiation therapy is selected from the form of electromagnetic waves, charged particles, or neutral particles.

4. The method of claim 1, wherein the radiation therapy is administered at a dose of about 60-70Gy over a period of 4-7 weeks.

5. The method of claim 1, wherein the solid tumor is selected from the group consisting of glioblastoma, squamous cell carcinoma, skin cancer-related tumors, breast cancer, head and neck cancer, gynecological cancer, urinary and male genital cancer, bladder cancer, prostate cancer, bone cancer, endocrine adenocarcinoma, digestive tract cancer, major digestive tract/organ cancer, central nervous system cancer, and lung cancer.

6. The method of claim 5, wherein the solid tumor is a glioblastoma.

7. The method of claim 1, wherein the solid tumor is a glioblastoma and the chemotherapy is temozolomide therapy.

8. The method according to claim 1, characterized in that crocetin is used as sensitizer.

9. The method of claim 1, further comprising administering with at least one anti-cancer treatment.

10. The method of claim 9, wherein the at least one anti-cancer treatment is selected from the group consisting of additional sensitizers, targeted therapeutics, and immunotherapeutics in the treatment of cancer.

11. The method of claim 9, wherein the at least one anticancer therapy is selected from the group consisting of anticancer alkylating agents, anticancer antimetabolites, anticancer antibiotics, plant-derived anticancer agents, anticancer platinum coordination compounds, anticancer camptothecin derivatives, anticancer tyrosine kinase inhibitors, monoclonal antibodies, biological response modifiers, and other anticancer agents.

12. The method according to claim 1, characterized in that an effective amount of crocetin is administered prior to the radiotherapy or chemotherapy or immunotherapy or combination thereof.

13. A composition comprising crocetin and a pharmaceutically acceptable carrier or adjuvant for use in the treatment of solid tumors.

14. The composition of claim 13, wherein the composition is an injection, a tablet, a capsule, a pill, a suppository, an aerosol, an oral liquid, a granule, a powder, a sustained release formulation, a nano-formulation, a syrup, a medicated wine, a tincture, a lotion, a film, or a combination thereof.

15. The composition of claim 13, wherein the composition is in the form of a liposomal formulation.

16. The composition of claim 13, wherein the composition is administered orally, by injection, by implantation, by spraying, by inhalation, or a combination thereof.

17. A method of sensitizing a mammal to radiation therapy, chemotherapy or a combination thereof comprising administering to the mammal a pharmaceutically effective amount of crocetin.

18. The method of claim 17, wherein the mammal is a human.